Chytrid Morphology Lab. Rabern Simmons University of Maine BIO432/532 Biology of the Fungi November 9-10, 2009

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1 Chytrid Morphology Lab Rabern Simmons University of Maine BIO432/532 Biology of the Fungi November 9-10, 2009

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3 Chytridiomycota Chytridiomycetes Things to see... Gross culture Different rhizoid types Endogenous vs. Exogenous Monocentric vs. Polycentric Sexual conjugation and resting spore formation Bd in bullfrog tadpole jaw sheath Monoblepharidomycetes Monoblepharis polymorpha Blastocladiomycota Allomyces sp.

4 Gross Culture Examination Lightly touch one side of the coverslip to the surface of the water Slowly lower the coverslip onto a slide Examine in a raster fashion at 40X objective until you see an epibiotic chytrid thallus

5 Chytrids in Gross Culture 1/10 mm

6 Chytrids in Gross Culture Endobiotic Epibiotic Empty zoosporangium 1/100 mm Mud under snow, Snowbird, Utah, July 26, 2009 Photos taken October 27, 2009

7 Pure Culture Examination Put a drop of water on a clean slide Flame scalpel & needle Cut small square with scalpel Scoop the very top of the agar as shallowly as possible Use needle to push agar off the scalpel into the water Slowly lower the coverslip onto a slide and add water to balance coverslip

8 Thallus Morphology Holocarpic Eucarpic Olpidium sp. (currently incertae sedis) Most common chytrid form. Sparrow, Aquatic Phycomycetes.

9 Holocarpic chytrid Zoosporangia with zoospore discharge tubes Plant cell wall Resting sporangia of fungus Olpidium brassicae in root cells (LM). Used with permission of D J S Barr.

10 Eucarpic chytrids Rhizoids Defining feature Robust and blunt-ended Sparse, fine and tapered Isodiametric... Just to name a few.

11 Rhizoids May have subsporangial swelling, or apophysis Chytriomyces aureus on chitin (Hoffman modulation contrast LM) JEL72 Unidentified Cladochytriales on agar (phase contrast LM) Unidentified Spizellomycetales on agar (LM)

12 Development of the Zoospore Cyst Endogenous Exogenous Zoospore cyst JEL369 Clydaea vesicula on agar (phase contrast LM) Barr81 Powellomyces hirtus on agar (Hoffman modulation contrast LM)

13 Endogenous Growth: Swelling of zoospore cyst Zoospore with flagellum Rhizoidal axis JEL539 Maunachytrium keaense on agar (phase contrast LM)

14 Exogenous Growth: Nuclear migration Zoospore cyst (zc) Zoosporangium zc zc Rhizoidal axis JEL129 Entophlyctis luteolus on agar (phase contrast LM)

15 Number of zoosporangia Monocentric Polycentric Zoosporangium Papilla Rhizoids Rhizomycelium Rhizoids JEL 95 Powellomyces sp. on agar (Hoffman modulation contrast LM) Zoosporangia JEL109 Polychytrium aggregatum on agar (phase contrast LM)

16 Polycentric Chytrids Rhizomycelium is coenocytic or rarely septate JEL109 Polychytrium aggregatum on agar (phase contrast LM) JEL610 Cladochytrium sp. on agar (phase contrast LM)

17 Polycentricity continued Terminal or intercalary zoosporangia & swellings JEL109 Polychytrium aggregatum on agar (phase contrast LM) M53 Monoblepharis sp. On agar (phase contrast LM) JEL611 Nowakowskiella sp. on agar (phase contrast LM)

18 More polycentrics on agar JEL137 Physocladia obscura JEL342 Catenomyces persicinus M36 Gonapodya polymorpha M6 Gonapodya prolifera

19 Resting Spore Formation Sexual conjugation produces resting spore Look for small thalli Larger thalli are more developed and conjugation is obscured Around the small thalli, you should see two small zoospore cysts that are empty Karyogamy of the two haploids in the resting spore

20 Batrachochytrium dendrobatidis In tadpoles, jaw sheath is the only keratinized tissue Infection by Bd causes discoloration Look for small, shiny spheres (the sheen of chitin cell wall), or tell-tale colonial thalli

21 Monoblepharidomycetes Almost all taxa are polycentric Invert plate at low objective magnification to observe growth within agar medium Look for oogonia and antheridia

22 Blastocladiomycota Allomyces sp. Hope to see gametangia Look for zoosporangia and resistant sporangia

23 Chytridiomycota Chytridiomycetes Things to see... Gross culture Different rhizoid types Endogenous vs. Exogenous Monocentric vs. Polycentric Sexual conjugation and resting spore formation Bd in bullfrog tadpole jaw sheath Monoblepharidomycetes Monoblepharis polymorpha Blastocladiomycota Allomyces sp.